Reciprocal expression of CD45RA and CD45RO in human CD4+ T cells defines populations understood to be naive cells (CD45RA+CD45RO-) and memory cells (CD45RA-CD45RO+). We investigate two subsets of CD45RA-CD45RO+ CD4+ human T cells which differ by fourfold in their expression of the CD45RB isoform; one is CD45RBbright and the other is CD45RBintermediate. In contrast, CD45RA+ naive cells are all CD45RBbright. Both subsets of CD45RA- cells proliferate in response to recall antigens so we designate them MEM 1 (CD45RO+RBbright) and MEM 2 (CD45RO+RBintermediate). CD45RA and CD45RB expression are regulated independently during in vitro activation of naive cells. When MEM 1 cells are activated they tend to down-regulate CD45RB expression, whereas activated MEM 2 cells tend to up-regulate CD45RB expression. Thus, in contrast to the stability of the CD45RA-CD45RO+ phenotype, the MEM 1 and MEM 2 phenotypes are labile and may interconvert. MEM 1 and MEM 2 cells produced comparable amounts of interleukin(IL)-2, IL-4, and IL-5 though MEM 1 cells produced slightly more interferon(IFN)-gamma (mean 1.7-fold more). MEM 1 cells consistently proliferated more (mean 2.3-fold more) than MEM 2 cells early during in vitro activation. Thus, differential expression of CD45RB within CD45RA- cells defines two subsets that have similar properties except for somewhat greater IFN-gamma production and proliferative responses by MEM 1 cells. Variability in CD45RB expression may represent a mechanism for fine-tuning the responsiveness of memory cells in vivo.